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SNP-mediated disruption of CTCF binding at the IFITM3 promoter is associated with risk of severe influenza in humans

Abstract

Previous studies have reported associations of IFITM3 SNP rs12252 with severe influenza, but evidence of association and the mechanism by which risk is conferred remain controversial. We prioritized SNPs in IFITM3 on the basis of putative biological function and identified rs34481144 in the 5′ UTR. We found evidence of a new association of rs34481144 with severe influenza in three influenza-infected cohorts characterized by different levels of influenza illness severity. We determined a role for rs34481144 as an expression quantitative trait locus (eQTL) for IFITM3, with the risk allele associated with lower mRNA expression. The risk allele was found to have decreased IRF3 binding and increased CTCF binding in promoter-binding assays, and risk allele carriage diminished transcriptional correlations among IFITM3-neighboring genes, indicative of CTCF boundary activity. Furthermore, the risk allele disrupts a CpG site that undergoes differential methylation in CD8+ T cell subsets. Carriers of the risk allele had reduced numbers of CD8+ T cells in their airways during natural influenza infection, consistent with IFITM3 promoting accumulation of CD8+ T cells in airways and indicating that a critical function for IFITM3 may be to promote immune cell persistence at mucosal sites.Our study identifies a new regulator of IFITM3 expression that associates with CD8+ T cell levels in the airways and a spectrum of clinical outcomes.

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Figure 1: IFITM3 regulatory SNP rs34481144 lies within the promoter region and is associated with clinical illness in three human cohorts.
Figure 2: Regulatory effect of rs34481144 genotype on IFITM3.
Figure 3: Identification of changes in protein binding to the IFITM3 promoter region due to rs34481144 genotype.
Figure 4: Genotype at rs34481144 alters methylation of the IFITM3 promoter, correlates with CD8+ T cell abundance in nasal washes, and results in broader effects on the locus.

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Acknowledgements

The authors thank the participants who contributed in each of the studies described in this manuscript. For FLU09, they acknowledge the clinical work of L. Harrison and the clinical team at the UTHSC, Le Bonheur Children's Hospital. They thank J.J. Treanor (University of Rochester Medical Center) for providing counsel regarding study design; R. Bajracharya, T.H. Oguin III, and L. McClaren for technical assistance; A. Webb, J.C. Crumpton, K. Friedman, T. Jeevan, and S.-S. Wong for their help with virological assays; and P. McKenzie and K. Johnson for assisting in the study management. They thank M. Weilnau, J. Parker, R. Eila, and R. Tabrizi for data management. They thank the Genentech FluA team for access to clinical samples. For PICFlu, they acknowledge the hard work and collaboration of the PALISI PICFlu Study Site Investigators who critically reviewed the initial study proposal and all modifications and enrolled and collected data on the patients in this study: Arkansas Children's Hospital, Little Rock, Arkansas, USA (R.C. Sanders, G. Hefley); Phoenix Children's Hospital, Phoenix, Arizona, USA (D. Tellez, C. Bliss, A. Labell, D. Liss, A.L. Ortiz); Children's Hospital of Los Angeles, Los Angeles, California, USA (B. Markovitz, J. Terry, R.S.P. Morzov); Children's Hospital Central California, Madera, California, USA (A.L. Graciano, M. Baldwin); Children's Hospital and Research Center, Oakland, California, USA (H. Flori, B. Brumfield, J. Simon); Children's Hospital of Orange County, Orange, California, USA (N. Anas, A. Schwarz, C. Onwunyi, S. Osborne, T. Patterson, O. Vargas-Shiraishi); UCSF Benioff Children's Hospital, University of California, San Francisco, San Francisco, California, USA (A. Sapru, M. Convery, V. Lo); Children's Hospital Colorado, Aurora, Colorado, USA (A. Czaja, P. Mourani, V. Batara Aymami, S. Burr, M. Brocato, S. Huston, E. Jewett, S.B. Lindahl, D. Loyola, Y. Sierra); Connecticut Children's Medical Center, Hartford, Connecticut, USA (C. Carroll, K.A. Sala, S. Thornton-Thompson); Yale–New Haven Children's Hospital, New Haven, Connecticut, USA (J.S. Giuliano Jr.); Holtz Children's Hospital, Miami, Florida, USA (G. McLaughlin); Children's Healthcare of Atlanta at Egleston, Atlanta, Georgia, USA (M. Paden, C.-C. Manghram, S. Meisner, C.L. Stone, R. Toney); University of Chicago Medicine Comer Children's Hospital, Chicago, Illinois, USA (J. Bubeck Wardenburg, A. DeDent); Kosair Children's Hospital, Louisville, Kentucky, USA (V. Montgomery, T. Evans, K. Richardson); Boston Children's Hospital, Boston, Massachusetts, USA (A. Randolph, A.A. Agan, E.M. Smith, R.M. Sullivan, G. Yoon, M. Kiers, S.M. Keisling); Johns Hopkins Children's Center, Baltimore, Maryland, USA (M. Bembea, E.D. White); Children's Hospital and Clinics of Minnesota, Minneapolis, Minnesota, USA (S.C. Kurachek, A.A. Doucette, E. Zielinski); St. Louis Children's Hospital, St. Louis, Missouri, USA (A. Doctor, M. Hartman, R. Jacobs); Children's Hospital of Nebraska, Omaha, Nebraska, USA (E. Truemper, M. Dawson); Children's Hospital at Dartmouth-Hitchcock, Lebanon, New Hampshire, USA (D.L. Levin, J.D. Jarvis); Children's Hospital at Montefiore, Bronx, New York, USA (C. Katyal); Golisano Children's Hospital, Rochester, New York, USA (K. Ackerman, L.E. Daugherty, L. Baglia); Nationwide Children's Hospital, Columbus, Ohio, USA (M.W. Hall, K. Greathouse, L. Steele); Penn State Children's Hospital, Hershey, Pennsylvania, USA (N. Thomas, J. Raymond, D. Spear); Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA (J. Fitzgerald, M. Helfaer, S. Weiss, J.L. Bush, M.A. Diliberto, J. Egan, B.B. Park, M. Sisko); Monroe Carell Jr. Children's Hospital at Vanderbilt, Nashville, Tennessee, USA (F.E. Barr, J. Arnold); Dell Children's Medical Center of Central Texas, Austin, Texas, USA (R. Higgerson, L. Christie); Children's Medical Center, Dallas, Texas, USA (M. Thompson); Texas Children's Hospital, Houston, Texas, USA (L.L. Loftis, N. Jaimon, U. Kyle); University of Virginia Children's Hospital, Charlottesville, Virginia, USA (D.F. Willson, C. Traul, R.L. Kelly); Children's Hospital of Wisconsin, Milwaukee, Wisconsin, USA (R. Gedeit, B.E. Horn, K. Luther, K. Murkowski); Centre Hospitalier Universitaire Sainte-Justine, Montreal, Quebec, Canada (P.A. Jouvet, A.-M. Fontaine); Centre Hospitalier de l'Université Laval, Quebec City, Quebec, Canada (M.-A. Dugas). This work was funded by the National Institute of Allergy and Infectious Diseases, US National Institutes of Health, under HHS contracts HHSN266200700005C and HHSN272201400006C for the St. Jude Center of Excellence for Influenza Research and Surveillance (P.G.T.), Genentech, Inc. (P.G.T., C.M.R.), the US National Institutes of Health (NIH AI084011 to A.G.R.), the Centers for Disease Control and Prevention (CDC; A.G.R.), and ALSAC. C.M.R., T.B., A.D., and M.O. are employees of Genentech, Inc.

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E.K.A., C.M.R., and P.G.T. wrote the manuscript and designed figures. E.K.A., A.G.R., T.B., P.D., C.M.O., B.Y., C.M.R., and P.G.T. designed experiments. E.K.A., P.D., M.O., and C.M.R. conducted experiments. C.M.O., A.E.Z., and J.P.S. acquired data. E.K.A., T.B., M.O., A.E.Z., D.F., A.D., C.M.R., and P.G.T. analyzed data. E.K.A., A.G.R., T.B., P.D., B.Y., C.M.R., and P.G.T. interpreted data. A.G.R. oversaw recruitment, data acquisition, and analysis for PICFlu. J.D. and M.C. oversaw recruitment for FLU09. A.G.R., T.B., P.D., M.O., and B.Y. edited the manuscript. All authors approved the final manuscript.

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Correspondence to Paul G Thomas.

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C.M.R., T.B., M.O., and A.D. are employees of Genentech, Inc.

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Allen, E., Randolph, A., Bhangale, T. et al. SNP-mediated disruption of CTCF binding at the IFITM3 promoter is associated with risk of severe influenza in humans. Nat Med 23, 975–983 (2017). https://doi.org/10.1038/nm.4370

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